HOXA3 Knockdown Impairs hESC-Derived Thymic Epithelial-like Cell Differentiation and CXCL12-Associated Early T-Lineage Support

Thymic epithelial cells (TECs) provide essential signals for T-cell development, yet the mechanisms governing human TEC differentiation and the acquisition of T-cell-supporting function remain incompletely understood. Using a three-stage system that directs human embryonic stem cells (hESCs) through definitive endoderm and third pharyngeal pouch endoderm (3PPE) toward a thymic epithelial-like lineage, we investigated the role of HOXA3 during thymic epithelial-like cell (TEC-like cell) differentiation. HOXA3 knockdown during 3PPE-to-TEC-like cell transition impaired thymic epithelial differentiation, as indicated by reduced K8, AIRE, and CD205 expression and decreased FOXN1 protein. HOXA3-knockdown TEC-like cells also showed a markedly reduced capacity to support the generation of CD45+CD3+ cells from cord blood CD34+ progenitors. Bulk RNA sequencing identified 1040 upregulated and 774 downregulated genes, revealing broad remodeling of cell-cycle, motility, chemotaxis, angiogenesis, and tissue-remodeling programs. CXCL12 was among the downregulated genes, and its reduction was confirmed at both mRNA and protein levels. Motif analysis identified a putative HOXA3-binding site, and conventional ChIP-PCR showed enrichment of the predicted CXCL12 regulatory region in HOXA3 immunoprecipitates. Recombinant CXCL12 significantly increased CD45+CD3+ cell generation in HOXA3-knockdown co-cultures. Together, these data support an association between HOXA3 knockdown, reduced acquisition of a thymic epithelial-like phenotype, and diminished early T-lineage support, with CXCL12 partially accounting for the functional phenotype. This study provides a tractable hESC-based model for studying human thymic epithelial development and TEC-mediated early T-lineage differentiation.